Vectors and methods for recombinant protein expression

ABSTRACT

There is disclosed an expression vector utilizing an internal polyadenylation signal. The internal polyadenylation signal is inserted between a DNA encoding a protein of interest and a DNA encoding a selectable marker, and allows a single promoter to generate both monocistronic messages and dicistronic messages. Similar, multicistronic vectors can also be prepared. Also disclosed are methods of using the expression vector utilizing an internal polyadenylation signal, host cells transfected therewith, stable pools of cells transfected with an expression vector utilizing an internal polyadenylation signal, and clones of such transfected cells.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to expression of recombinantproteins in eukaryotic cells.

BACKGROUND OF THE INVENTION

[0002] The development of expression systems for production ofrecombinant proteins is important for providing a source of a givenprotein for research or therapeutic use. Expression systems have beendeveloped for both prokaryotic cells, such as E. coli, and foreukaryotic cells, such as yeast (i.e., Saccharomyces, Pichia andKluyveromyces spp) and mammalian cells. Expression in mammalian cells isoften preferred for manufacturing of therapeutic proteins, sincepost-translational modifications in such expression systems are morelikely to resemble those occurring on endogenous proteins in a mammal,than the type of post-translational modifications that occur inmicrobial expression systems.

[0003] Several vectors are available for expression in mammalian hosts,each containing various combinations of cis- and in some casestrans-regulatory elements to achieve high levels of recombinant proteinin a minimal time frame. However, despite the availability of numeroussuch vectors, the level of expression of a recombinant protein achievedin mammalian systems is often lower than that obtained with a microbialexpression system. Additionally, because only a small percentage ofcloned, transfected mammalian cells express high levels of the proteinof interest, it can often take a considerably longer time to developuseful stably transfected mammalian cell lines than it takes formicrobial systems.

[0004] The use of a dicistronic expression vector wherein a first openreading frame encodes a polypeptide of interest and a second openreading frame encodes a selectable marker, is one method that has beenused to obtain recombinant proteins. A preferred marker for use in suchsystems is dihydrofolate reductase (DHFR), which has the advantage ofbeing an amplifiable gene, allowing selection for cells having high copynumbers of the inserted DNA by culturing them in increasing levels ofmethotrexate (MTX). However, translation of the selectable marker geneis up to 100-fold less efficient than translation of the gene ofinterest, which reduces the efficiency of the selection process.Moreover, dicistronic expression vectors tend to undergo deletion orrearrangement under amplification conditions, in an uncontrolled manner,increasing the chances that amplified cells will no longer express theprotein of interest.

[0005] Internal ribosome entry sites (IRES) are a type of regulatoryelement found in several viruses and cellular RNAs (reviewed inMcBratney et. al. Current Opinion in Cell Biology 5:961, 1993). IRESincrease the efficiency of translation of the selectable marker gene,and are thus useful in enhancing both the selection and amplificationprocess (Kaufman R. J., et al., Nucleic Acids Res. 19:4485, 1991).Nonetheless, the available evidence indicates that dicistronic mRNAsaccumulate to lower levels than monocistronic mRNAs, possibly because ofreduced mRNA stability of the longer message.

[0006] Because the amount of recombinant protein produced by atransfected cell is generally proportional to the amount of mRNAavailable for translation of the protein, the use of dicistronicexpression vectors may result in low levels of production of the desiredrecombinant protein. Accordingly, there is a need in the art to developimproved methods that retain the utility of a selectable, amplifiablemarker such as DHFR, while increasing the proportion of mRNAs encodingthe desired recombinant protein. Moreover, there is a need to developmethods that facilitate selection of those transfectants that integrateinto more transcriptionally active sites, and that allow production ofuseful levels of recombinant protein from mammalian cells in arelatively short period of time.

SUMMARY OF THE INVENTION

[0007] In one embodiment of the invention, an expression vectorcomprises a DNA encoding a first protein, operably linked to a DNAencoding a second protein, wherein a DNA encoding a polyadenylation(polyA) site is inserted between the DNA encoding the first protein ofinterest and the DNA encoding the second protein, such that the DNAencoding the internal polyadenylation site is operably linked to the DNAencoding the first. A preferred second protein is selectable marker,preferably dihydrofolate reductase (DHFR); other amplifiable markers arealso suitable for use in the inventive expression vectors.

[0008] Preferably, the polyadenylation signal utilized to provide theinternal polyadenylation site is an SV40 polyadenylation signal, morepreferably, the late SV40 polyadenylation signal, and most preferably, amutant version of the late SV40 polyadenylation signal. The preferredpolyadenylation signals are presented in the Sequence Listing anddescribed further below. In another embodiment of the invention, thepolyadenylation signal is inducible.

[0009] The expression vector may further comprise an IRES sequencebetween the DNA encoding the first protein, and the DNA encoding thesecond protein, operably linked to both and downstream of the internalpolyadenylation site. Alternatively, the expression vector may comprisemRNA splice donor and acceptor sites substantially as described by Lucaset al. infra.

[0010] Another aspect of the invention comprises an expression vectorinto which a DNA encoding a protein. Such an expression vector comprisesa site into which a DNA encoding a recombinant, heterologous protein canbe inserted (referred to as a cloning site), such that it is operablylinked to an internal polyadenylation site and a DNA encoding a secondprotein (such as a selectable marker). Optionally, other regulatoryelements may also be included, for example, an IRES sequence downstreamof the internal polyadenylation site, or mRNA splice donor and acceptorsites substantially as described by Lucas et al. infra, operably linkedto the internal polyadenylation site and the DNA encoding the secondprotein. An expression-augmenting sequence element (EASE) may also beincluded upstream of the cloning site, operably linked thereto.

[0011] Host cells can be transfected with the inventive expressionvectors, yielding stable pools of transfected cells. Accordingly,another embodiment of the invention provides a transfected host cell;yet another embodiment provides a stable pools of cells transfected withthe inventive expression vector. Also provided are cell lines clonedfrom pools of transfected cells. Preferred host cells are mammaliancells. In a most preferred embodiment, the host cells are CHO cells.

[0012] The invention also provides a method for obtaining a recombinantprotein, comprising transfecting a host cell with an inventiveexpression vector, culturing the transfected host cell under conditionspromoting expression of the protein, and recovering the protein. In apreferred application of this invention, transfected host cell lines areselected with two selection steps, the first to select for cellsexpressing the dominant amplifiable marker, and the second step for highexpression levels and/or amplification of the marker gene as well as thegene of interest. In a most preferred embodiment, the selection oramplification agent is methotrexate, an inhibitor of DHFR that has beenshown to cause amplification of endogenous DHFR genes and transfectedDHFR sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a diagram of constructs prepared in Example 1. Theconstruct in which the SV40 early polyadenylation signal was includedwas designated SPA6; that in which the late polyadenylation signal wasincluded was designated SPA4. A control construct was designated BGH.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Expression vectors that retain the utility of a selectable,amplifiable marker such as DHFR, while increasing the proportion ofmRNAs encoding a desired recombinant protein, are provided herein. Theinventive expression vectors comprise a polyadenylation signal insertedbetween a first coding sequence and a second or subsequent codingsequence (referred to as an internal polyadenylation site). In theinventive vectors, transcripts originating at the promoter can bepolyadenylated following the first coding sequence (monocistronicmessage) or after the second or subsequent coding sequence(multicistronic message). In one embodiment of the invention, the firstcoding sequence encodes a protein of interest, and the second (orsubsequent) coding sequence encodes a selectable marker. In anotherembodiment, a second polyadenylation site follows the second orsubsequent coding sequence, and is operably linked thereto. In thisembodiment, the internal polyadenylation site thus becomes the firstpolyadenylation site.

[0015] Because many transcripts encode only the gene of interest and notthe selectable marker, the inventive vectors produce less selectablemarker protein, and only those transfectants that integrate into moretranscriptionally active sites survive the selection process.Accordingly, use of the inventive expression vectors facilitatesisolation of transfected pools and clones that express high levels ofrecombinant protein using lower levels of a selection agent than ispossible in the absence of the internal polyadenylation signal.

[0016] An additional benefit of utilizing the inventive expressionvectors is that monocistronic messages may be more stable or moreefficiently processed than dicistronic messages, potentially leading toincreased accumulation of the message encoding the protein of interest,and hence to higher levels of protein production. Use of the inventiveinternal polyadenylation site will thus facilitate production of usefullevels of recombinant protein by transfected cells in a relatively shortperiod of time.

[0017] The inventive vectors and methods will also be useful indeveloping multicistronic vectors. Multicistronic expression vectorsallow the coordinated expression of two or more genes (see, for example,Fussenegger et al., Biotechnol Prog 13:733; 1997). Inserting apolyadenylation site after a first cistron would result in high levelexpression of the first cistron and lower level expression of anyfollowing cistrons. Potential applications of this technology would beto facilitate expression of large amounts of a therapeutic protein (orother, desired recombinant proteins) and lower amounts of other proteinssuch as selectable markers, transcription factors, enzymes involved inprotein folding, and other proteins that regulate cell metabolism andexpression.

[0018] In another embodiment, the polyadenylation site is inserted afterthe second or third (or subsequent) cistron. This would allow highexpression of the first two (or three or more) cistrons, followed bylower expression of the cistron following the internal polyadenylationsite. This embodiment will find use, for example, in recombinantantibody synthesis where the heavy and light chains are synthesizedindependently at high levels. A tricistronic vector is constructed withthe heavy and light chains encoded by the first two cistrons. Thepolyadenlylation site is inserted following the second cistron allowinghigh level expression of the first two cistrons. The selectable markeris expressed from the third cistron (i.e., after the polyadenylationsite) and would be expressed at lower levels.

[0019] Expression of Recombinant Proteins

[0020] As used herein, the term ‘expression vector’ is understood todescribe a vector that comprises various regulatory elements, describedin detail below, that are necessary for the expression of recombinant,heterologous proteins in cells. The expression vector can includesignals appropriate for maintenance in prokaryotic or eukaryotic cells,and/or the expression vector can be integrated into a chromosome.

[0021] Recombinant expression vectors may include a coding sequenceencoding a protein of interest (or fragment thereof), ribozymes,ribosomal mRNAs, antisense RNAs and the like. Preferably, the codingsequence encodes a protein or peptide. The coding sequence may besynthetic, a cDNA-derived nucleic acid fragment or a nucleic acidfragment isolated by polymerase chain reaction (PCR). The codingsequence is operably linked to suitable transcriptional or translationalregulatory elements derived from mammalian, viral or insect genes. Suchregulatory elements include a transcriptional promoter, a sequenceencoding suitable mRNA ribosomal binding sites, and sequences whichcontrol the termination of transcription and translation (i.e., apolyadenylation signal), as described in detail below.

[0022] Expression vectors may also comprise non-transcribed elementssuch as a suitable promoter and/or enhancer linked to the gene to beexpressed, other 5′ or 3′ flanking non-transcribed sequences, 5′ or 3′non-translated sequences such as ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. An origin of replication thatconfers the ability to replicate in a host, and a selectable gene tofacilitate recognition of transfectants, may also be incorporated.

[0023] DNA regions are operably linked when they are functionallyrelated to each other. For example, DNA for a signal peptide (secretoryleader) is operably linked to DNA for a polypeptide if it is expressedas a precursor which participates in the secretion of the polypeptide;thus, in the case of DNA encoding secretory leaders, operably linkedmeans contiguous and in reading frame. A promoter is operably linked toa coding sequence if it controls the transcription of the sequence; anda ribosome binding site is operably linked to a coding sequence if it ispositioned so as to permit translation.

[0024] Dicistronic expression vectors used for the expression ofmultiple transcripts have been described previously (Kim S. K. and WoldB. J., Cell 42:129, 1985; Kaufman et al. 1991, supra). Dicistronicexpression vectors comprise two cistrons, or open reading frames,capable of encoding two proteins, for example, a recombinant of interestand a selectable marker. An example of such dicistronic expressionvector is pCAVDHFR, a derivative of pCD302 (Mosley et al., Cell 1989)containing the coding sequence for mouse DHFR (Subramani et al., Mol.Cell. Biol. 1:854, 1981). Another example of such distronic expressionvector is pCDE vector, a derivative of pCAVDHFR containing the murineencephalomyocarditis virus internal ribosomal entry site (nucleotides260 through 824; Jang and Wimmer, Genes and Dev. 4:1560, 1990) clonedbetween the adenovirus tripartite leader and the DHFR cDNA codingsequence. Other types of expression vectors will also be useful incombination with the invention, for example, those described in U.S.Pat. No. 4,634,665 (Axel et al.) and U.S. Pat. No. 4,656,134 (Ringold etal.).

[0025] The transcriptional and translational control sequences inexpression vectors to be used in transfecting cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. Viral genomic promoters, control and/or signalsequences may be utilized to drive expression, provided such controlsequences are compatible with the host cell chosen. Examples of suchvectors can be constructed as disclosed by Okayama and Berg (Mol. Cell.Biol. 3:280, 1983). Non-viral cellular promoters can also be used (i.e.,the beta-globin and the EF-1alpha promoters), depending on the cell typein which the recombinant protein is to be expressed.

[0026] DNA sequences derived from the SV40 viral genome, for example,SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al., Nature 273:113, 1978). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind III site toward the BglI site locatedin the viral origin of replication is included.

[0027] In dicistronic expression vectors, a polyadenylation siteinserted downstream of, and operably linked to, the second cistron(usually, a DNA encoding a selectable marker), is often used to regulatetranscription and translation. Many such polyadenylation signal areknown (see for example, Table 1 below). The present invention utilizesan internal polyadenylation signal, eg., one that is inserted betweenthe two cistrons of a dicistronic expression vector, in addition to apolyadenylation signal or other suitable regulatory element downstreamof the second cistron.

[0028] Both the early and late polyadenylation signals of SV40 areuseful in the instant invention. These sequences are encoded within the237-base pair fragment between the BamHI site at nucleotide 2533 and theBclI site at nucleotide 2770 of the SV40 genome (Carswell and Alwine,Mol. Cell. Biol. 9:4248; 1989). Carswell and Alwine concluded that, ofthe two SV40 polyadenylation signals, the late signal was moreefficient, most likely because it comprises both downstream and upstreamsequence elements that facilitate efficient cleavage andpolyadenylation.

[0029] Many polyadenylation signals are known in the art, and will alsobe useful in the instant invention. Examples include those shown inTable 1 below. TABLE 1 Polyadenylation Signals SV40 late polyA Schek, N,Cooke, C., and J. C. Alwine (1992): and deletion Definition of theupstream efficiency element of the mutants simian virus 40 latepolyadenylation signal by using thereof in vitro analysis. Mol. CellBiol. 12:5386-5393 HIV-1 polyA Klasens, B. I. F., Das, A. T., and B.Berkhout (1998): Inhibition of polyadenylation by stable RNA secondarystructure. Nucleic Acids Res. 26:1870-1876 β-globin polyA Gil, A., andN. J. Proudfoot. (1987): Position- dependent sequence elementsdownstream of AAUAAA are required for efficient rabbit β-globin mRNAformation. Cell 49:399-406 HSV TK polyA Cole, C. N. and T. P. Stacy(1985): Identification of sequence in the herpes simplex virus thymidinekinase gene required for efficient processing and poly- adenylation.Mol. Cell. Biol. 5:2104-2113 Polyomavirus Batt, D. B and G. G.Carmichael (1995): polyA Characterization of the polyomavirus late poly-adenylation signal. Mol. Cell. Biol. 15:4783-4790 Bovine growth Gimmi,E. R., Reff, M. E., and I. C. Deckman. hormone (1989): Alterations inpre-mRNA topology of the bovine growth hormone polyadenylation regiondecrease polyA site efficiency. Nucleic Acids Res. 17:6983-6998

[0030] Additional polyadenylation sites can be identified or constructedusing methods that are known in the art. A minimal polyadenylation siteis composed of AAUAAA and a second recognition sequence, generally a G/Urich sequence, found about 30 nucleotides downstream. In the SequenceListing, the sequences are presented as DNA, rather than RNA, tofacilitate preparation of suitable DNAs for incorporation intoexpression vectors. When presented as DNA, the polyadenylation site iscomposed of AATAAA, with, for example, a G/T rich region downstream (seefor example, nucleotides 123 through 128 and 151 through 187,respectively, of SEQ ID NO:1).

[0031] Both sequences must be present to form an efficientpolyadenylation site. The purpose of these sites is to recruit specificRNA binding proteins to the RNA. The AAUAAA binds cleavagepolyadenylation specificity factor (CPSF; Murthy K. G., and Manley J. L.(1995), Genes Dev 9:2672-2683), and second site, frequently a G/Usequence, binds to Cleavage stimulatory factor (CstF; Takagaki Y. andManley J. L. (1997) Mol Cell Biol 17:3907-3914). CstF is composed ofseveral proteins, but the protein responsible for RNA binding isCstF-64, a member of the ribonucleoprotein domain family of proteins(Takagaki et al. (1992) Proc Natl Acad Sci USA 89:1403-1407).

[0032] The concentration of CstF-64 protein has been shown to beimportant in regulating usage of different polyadenylation sites inB-cells (Takagaki Y, Manley J L (1998) Mol Cell 2:761-771) Accordingly,an inducible polyadenylation site can be constructed based on thisnaturally occurring regulation of polyadenylation usage in B-cell, bycontrolling the interaction of CstF-64 with an mRNA of choice to inducepolyadenylation. For example, the CstF-64 may be fused to the RNAbinding domain of the MS2 phage coat protein, which binds a specific RNAsequence (ACAUGAGGAUUACCCAUGU; SEQ ID NO:4) distinct from the G/U richelement (Lowary and Uhlenbeck (1987) Nucleic Acids Res. 15:10483+10493).The target mRNA would contain an AAUAAA sequence and an MS2 coat proteinRNA recognition sequence. By regulating the level of the MS2-CstF-64fusion protein transcriptionally using standard inducible expressionsystems (for example, an Ecdysone-inducible mammalian expression systemdescribed by No et al. (1996) Proc Natl Acad Sci USA 93:3346-3351), theusage of the inducible polyA site could be controlled.

[0033] Polyadenylation may also be regulated by developingtemperature-sensitive MS2 RNA binding domain mutants. MS2 RNA bindingdomain mutants may be generated using random mutagenesis, and screenedfor temperature sensitivity. When used as a fusion partner with CstF-64as described above, the temperature-sensitive MS2 coat protein would beinactive and fail to bind RNA at 37° C.; thus the internal polyA sitewould not function at this temperature. However, at reduced temperature,for example 32° C., the MS2 coat protein would be active, wouldrecognize the RNA sequence target, and the message would bepolyadenylated. Temperature regulation would be particularly useful forrecombinant protein expression, since reducing the temperature ofexpression cultures is typically used to increase protein expression.

[0034] An additional technique that can be used in conjunction with theinventive vectors is described by Lucas et al. (Nucleic Acids Res.24:1774; 1996). In an effort to increase production of a desiredprotein, Lucas et al. utilized mRNA splice donor and acceptor sites todevelop stable clones that produced both a selectable marker andrecombinant proteins. According to these investigators, the vectors theyprepared resulted in the transcription of a high proportion of mRNAencoding the desired protein, and a fixed, relatively low level of theselection marker that allowed selection of stable transfectants.

[0035] Host Cells

[0036] Transfected host cells are cells which have been transfected(sometimes referred to as ‘transformed’) with heterologous DNA. Manytechniques for transfecting cells are known; in one approach, cells aretransfected with expression vectors constructed using recombinant DNAtechniques and which contain sequences encoding recombinant proteins.Expressed proteins will preferably be secreted into the culturesupernatant, but may be associated with the cell membrane, depending onthe particular polypeptide that is expressed. Mammalian host cells arepreferred for the instant invention. Various mammalian cell culturesystems can be employed to express recombinant protein. Examples ofsuitable mammalian host cell lines include the COS-7 lines of monkeykidney cells, described by Gluzman (Cell 23:175, 1981), CV-1/EBNA (ATCCCRL 10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa andBHK cell lines.

[0037] A commonly used cell line is DHFR− CHO cells which areauxotrophic for glycine, thymidine and hypoxanthine, and can betransformed to the DHFR+ phenotype using DHFR cDNA as an amplifiabledominant marker. One such DHFR− CHO cell line, DXB11, was described byUrlaub and Chasin (Proc. Natl. Acad. Sci. USA 77:4216, 1980). Anotherexample of a DHFR− CHO cell line is DG44 (see, for example, Kaufman, R.J., Meth. Enzymology 185:537 (1988)). Other cell lines developed forspecific selection or amplification schemes will also be useful with theinvention.

[0038] Numerous other eukaryotic cells will also be useful in thepresent invention, including cells from other vertebrates, and insectcells. Those of skill in the art will be able to select appropriatevectors, regulatory elements, transfection and culture schemes accordingto the needs of their preferred culture system.

[0039] Preparation of Transfected Mammalian Cells

[0040] Several transfection protocols are known in the art, and arereviewed in Kaufman, R. J., supra. The transfection protocol chosen willdepend on the host cell type and the nature of the protein of interest,and can be chosen based upon routine experimentation. The basicrequirements of any such protocol are first to introduce DNA encodingthe protein of interest into a suitable host cell, and then to identifyand isolate host cells which have incorporated the heterologous DNA in astable, expressible manner.

[0041] One commonly used method of introducing heterologous DNA iscalcium phosphate precipitation, for example, as described by Wigler etal. (Proc. Natl. Acad. Sci. USA 77:3567, 1980). DNA introduced into ahost cell by this method frequently undergoes rearrangement, making thisprocedure useful for cotransfection of independent genes.

[0042] Polyethylene-induced fusion of bacterial protoplasts withmammalian cells (Schaffner et al., Proc. Natl. Acad. Sci. USA 77:2163,1980) is another useful method of introducing heterologous DNA.Protoplast fusion protocols frequently yield multiple copies of theplasmid DNA integrated into the mammalian host cell genome. Thistechnique requires the selection and amplification marker to be on thesame plasmid as the gene of interest.

[0043] Electroporation can also be used to introduce DNA directly intothe cytoplasm of a host cell, as described by Potter et al. (Proc. Natl.Acad. Sci. USA 81:7161, 1988) or Shigekawa and Dower (BioTechniques6:742, 1988). Unlike protoplast fusion, electroporation does not requirethe selection marker and the gene of interest to be on the same plasmid.

[0044] More recently, several reagents useful for introducingheterologous DNA into a mammalian cell have been described. Theseinclude Lipofectin® Reagent and Lipofectamine™ Reagent (Gibco BRL,Gaithersburg, Md.). Both of these reagents are commercially availablereagents used to form lipid-nucleic acid complexes (or liposomes) which,when applied to cultured cells, facilitate uptake of the nucleic acidinto the cells.

[0045] Transfection of cells with heterologous DNA and selection forcells that have taken up the heterologous DNA and express the selectablemarker results in a pool of transfected cells. Individual cells in thesepools will vary in the amount of DNA incorporated and in the chromosomallocation of the transfected DNA. After repeated passage, poolsfrequently lose the ability to express the heterologous protein. Togenerate stable cell lines, individual cells can be isolated from thepools and cultured (a process referred to as cloning), a laborious timeconsuming process. However, in some instances, the pools them selves maybe stable (i.e., production of the heterologous recombinant proteinremains stable). The ability to select and culture such stable pools ofcells would be desirable as it would allow rapid production ofrelatively large amounts of recombinant protein from mammalian cells.

[0046] A method of amplifying the gene of interest is also desirable forexpression of the recombinant protein, and typically involves the use ofa selection marker (reviewed in Kaufman, R. J., supra). Resistance tocytotoxic drugs is the characteristic most frequently used as aselection marker, and can be the result of either a dominant trait(i.e., can be used independent of host cell type) or a recessive trait(i.e., useful in particular host cell types that are deficient inwhatever activity is being selected for). Several amplifiable markersare suitable for use in the inventive expression vectors (for example,as described in Maniatis, Molecular Biology: A Laboratory Manual, ColdSpring Harbor Laboratory, NY, 1989; pgs 16.9-16.14).

[0047] Useful selectable markers for gene amplification indrug-resistant mammalian cells are shown in Table 1 of Kaufman, R. J.,supra, and include DHFR-MTX resistance, P-glycoprotein and multiple drugresistance (MDR)-various lipophilic cytoxic agents (i.e., adriamycin,colchicine, vincristine), and adenosine deaminase (ADA)-Xyl-A oradenosine and 2′-deoxycoformycin. Specific examples of genes that encodeselectable markers are those that encode antimetabolite resistance suchas the DHFR protein, which confers resistance to methotrexate (Wigler etal., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); the GPT protein, which confersresistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl.Acad. Sci. USA 78:2072), the neomycin resistance marker, which confersresistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981,J. Mol. Biol. 150:1); the Hygro protein, which confers resistance tohygromycin (Santerre et al., 1984, Gene 30:147); and the Zeocin™resistance marker (available commercially from Invitrogen). In addition,the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- and aprt-cells, respectively.

[0048] Other dominant selectable markers include microbially derivedantibiotic resistance genes, for example neomycin, kanamycin orhygromycin resistance. However, these selection markers have not beenshown to be amplifiable (Kaufman, R. J., supra,). Several suitableselection systems exist for mammalian hosts (Maniatis supra, pgs16.9-16.15). Co-transfection protocols employing two dominant selectablemarkers have also been described (Okayama and Berg, Mol Cell Biol5:1136, 1985).

[0049] A particularly useful selection and amplification scheme utilizesDHFR-MTX resistance. MTX is an inhibitor of DHFR that has been shown tocause amplification of endogenous DHFR genes (Alt F. W., et al., J BiolChem 253:1357, 1978) and transfected DHFR sequences (Wigler M., et al.,Proc. Natl. Acad. Sci. USA 77:3567, 1980). Cells are transfected withDNA comprising the gene of interest and DNA encoding DHFR in adicistronic expression unit (Kaufman et al., 1991 supra and Kaufman R.J., et al., EMBO J. 6:187, 1987). Transfected cells are grown in mediacontaining successively greater amounts of MTX, resulting in greaterexpression of the DHFR gene, as well as the gene of interest.

[0050] Useful regulatory elements, described previously, can also beincluded in the plasmids or expression vectors used to transfectmammalian cells. The transfection protocol chosen, and the elementsselected for use therein, will depend on the type of host cell used.Those of skill in the art are aware of numerous different protocols andhost cells, and can select an appropriate system for expression of adesired protein, based on the requirements of their selected cellculture system(s).

[0051] Uses of the Invention

[0052] The inventive vectors and methods will find use for theexpression of a wide variety of recombinant polypeptides. Examples ofsuch polypeptides include cytokines and growth factors, such asInterleukins 1 through 18, the interferons, RANTES, lymphotoxin-β, Fasligand, flt-3 ligand, ligand for receptor activator of NF-kappa B(RANKL), TNF-related apoptosis-inducing ligand (TRAIL), CD40 ligand,Ox40 ligand, 4-1BB ligand (and other members of the TNF family), thymicstroma-derived lymphopoietin, granulocyte colony stimulating factor,granulocyte-macrophage colony stimulating factor, mast cell growthfactor, stem cell growth factor, epidermal growth factor, growthhormone, tumor necrosis factor, leukemia inhibitory factor,oncostatin-M, and hematopoietic factors such as erythropoietin andthrombopoietin.

[0053] Also included are neurotrophic factors such as brain-derivedneurotrophic factor, ciliary neurotrophic factor, glial cell-linederived neurotrophic factor and various ligands for cell surfacemolecules Elk and Hek (such as the ligands for eph-related kinases, orLERKS). Descriptions of proteins that can be expressed according to theinventive methods may be found in, for example, Human Cytokines:Handbook for Basic and Clinical Research, Vol. II (Aggarwal andGutterman, eds. Blackwell Sciences, Cambridge Mass., 1998); GrowthFactors:A Practical Approach (McKay and Leigh, eds., Oxford UniversityPress Inc., New York, 1993) and The Cytokine Handbook (A W Thompson,ed.; Academic Press, San Diego Calif.; 1991).

[0054] Receptors for any of the aforementioned proteins may also beexpressed using the inventive vectors and methods, including both formsof tumor necrosis factor receptor (referred to as p55 and p75),Interleukin-1 receptors (type 1 and 2), Interleukin-4 receptor,Interleukin-15 receptor, Interleukin-17 receptor, Interleukin-18receptor, granulocyte-macrophage colony stimulating factor receptor,granulocyte colony stimulating factor receptor, receptors foroncostatin-M and leukemia inhibitory factor, receptor activator ofNF-kappa B (RANK), receptors for TRAIL, and receptors that comprisedeath domains, such as Fas or Apoptosis-Inducing Receptor (AIR).

[0055] Other proteins that can be expressed using the inventive vectorsand methods include cluster of differentiation antigens (referred to asCD proteins), for example, those disclosed in Leukocyte Typing VI(Proceedings of the VIth International Workshop and Conference;Kishimoto, Kikutani et al., eds.; Kobe, Japan, 1996), or CD moleculesdisclosed in subsequent workshops. Examples of such molecules includeCD27, CD30, CD39, CD40; and ligands thereto (CD27 ligand, CD30 ligandand CD40 ligand). Several of these are members of the TNF receptorfamily, which also includes 41BB and OX40; the ligands are often membersof the TNF family (as are 4-1BB ligand and OX40 ligand); accordingly,members of the TNF and TNFR families can also be expressed using thepresent invention.

[0056] Proteins that are enzymatically active can also be expressedaccording to the instant invention. Examples includemetalloproteinase-disintegrin family members, various kinases (includingstreptokinase and tissue plasminogen activator as well as DeathAssociated Kinase Containing Ankyrin Repeats, and IKR 1 and 2),TNF-alpha Converting Enzyme, and numerous other enzymes. Ligands forenzymatically active proteins can also be expressed by applying theinstant invention.

[0057] The inventive vectors and methods are also useful for expressionof other types of recombinant proteins, including immunoglobulinmolecules or portions thereof, and chimeric antibodies (i.e., anantibody having a human constant region couples to a murine antigenbinding region) or fragments thereof. Numerous techniques are known bywhich DNA encoding immunoglobulin molecules can be manipulated to yieldDNAs capable of encoding recombinant proteins such as single chainantibodies, antibodies with enhanced affinity, or other antibody-basedpolypeptides (see, for example, Larrick et al., Biotechnology 7:934-938,1989; Reichmann et al., Nature 332:323-327, 1988; Roberts et al., Nature328:731-734, 1987; Verhoeyen et al., Science 239:1534-1536, 1988;Chaudhary et al., Nature 339:394-397, 1989).

[0058] Various fusion proteins can also be expressed using the inventivemethods and vectors. Examples of such fusion proteins include proteinsexpressed as fusion with a portion of an immunoglobulin molecule,proteins expressed as fusion proteins with a zipper moiety, and novelpolyfunctional proteins such as a fusion proteins of a cytokine and agrowth factor (i.e., GM-CSF and IL-3, MGF and IL-3). WO 93/08207 and WO96/40918 describe the preparation of various soluble oligomeric forms ofa molecule referred to as CD40L, including an immunoglobulin fusionprotein and a zipper fusion protein, respectively; the techniquesdiscussed therein are readily applicable to other proteins.

[0059] As additional examples, DNAs based on one or more expressedsequence tag (EST) from a library of ESTs can be prepared, inserted intothe inventive vector and expressed to obtain recombinant polypeptide.Moreover, DNAs isolated by use of ESTs (i.e., by PCR or the applicationof other cloning techniques) can also be expressed by applying theinstant invention. Information on the aforementioned polypeptides, aswell as many others, can be obtained from a variety of public sources,including electronic databases such as GenBank. A particularly usefulsite is the website of the National Center for BiotechnologyInformation/National Library of Medicine/National Institutes of Health(www.ncbi.nlm.nih.gov). Those of ordinary skill in the art are able toobtain information needed to express a desired polypeptide and apply thetechniques described herein by routine experimentation.

[0060] However, for purposes of this application, the definition of aprotein of interest excludes genes encoding proteins that are typicallyused as selectable markers in cell culture such as auxotrophic,antimetabolite and/or antibiotic markers. Nevertheless, the inventiondoes include the use of a selectable marker as an aid in selecting cellsand/or amplifying clones that are genetically engineered to express agene of interest. Preferably, the selectable marker gene is positionedadjacent to the gene of interest such that selection and/oramplification of the marker gene will select and/or amplify the adjacentgene.

[0061] The relevant disclosures of all references cited herein arespecifically incorporated by reference. The following examples areintended to illustrate particular embodiments, and not limit the scope,of the invention. Those of ordinary skill in the art will readilyrecognize that additional embodiments are encompassed by the invention.

EXAMPLES Example 1

[0062] This example describes the preparation of several expressionvectors for the expression of a soluble form of a receptor for humanInterleukin-4, referred to as sIL-4R. Human IL-4R cDNA and protein aredisclosed in U.S. Pat. No. 5,840,869, issued Nov. 24, 1998; U.S. Pat.No. 5,599,905, issued Feb. 4, 1997 and U.S. Pat. No. 5,856,296, issuedJan. 5, 1999. SEQ ID NOs:5 and 6 present the nucleotide and amino acidsequence (respectively) of human IL4R. Amino acids −25 through −1comprise a putative leader peptide; cleavage has been found to occurbetween amino acids −1 and 1, and between amino acids −3 and −2. Aminoacids 208 through 231 form a transmembrane region. DNA encoding sIL-4Rfrom amino acid −25 to amino acid 207 was used in the expressionvectors.

[0063] The original expression vector, pCAVDHFR is a derivative ofpCD302 (Mosley et al., Cell 89:335-348; 1989) containing the codingsequence for mouse DHFR (Subramani et al., Mol. Cell. Biol. 1:854,1981). The pCDE vector is a derivative of pCAVDHFR containing the murineencephalomyocarditis virus IRES (nucleotides 260 through 824; Jang andWimmer, Genes and Dev. 4:1560, 1990) cloned between the adenovirustripartite leader and the DHFR cDNA coding sequence. Anexpression-augmenting sequence element (EASE) was included upstream ofthe CMV leader. The EASE is described in U.S. Pat. No. 6,027,915, issuedFeb. 22, 2000, and in U.S. Ser. No. 09/435,377, filed Nov. 5, 1999, nowallowed.

[0064] To allow polyadenylation of the dicistronic message, the bovinegrowth hormone polyadenylation site was placed 3′ of the DHFR gene. Theplasmid pBGH is a standard dicistronic vector and serves as the control.The alternate polyadenylation vectors of the present invention wereconstructed by inserting various polyadenylation sites between the IL-4Rand the IRES. The plasmids pSPA4, pSPA6, and pMLPA were constructed byinserting the late SV40 polyA site, the early SV40 polyA site, and adeletion mutant of the late SV40 polyA site, respectively. The deletionmutant late SV40 polyA site was constructed using PCR to isolate afragment of the late SV40 polyA, nucleotides 80 through 222 of SEQ IDNO: 1. A diagram of the various constructs is shown in FIG. 1; thenucleotide sequences of the various polyA sites are shown in theSequence Listing (SV40 late: SEQ ID NO:1; BGH: SEQ ID NO:2; SV40 early:SEQ ID NO:3).

[0065] The plasmids were used in standard transfections to preparetransfected cells expressing IL-4R. Dihydrofolate reductase (DHFR)deficient Chinese hamster ovary (CHO) cells DXB11 (Chasin and Urlaub,supra) cells were adapted to a DMEM:F12 based serum free mediumsupplemented with 2 mM L-glutamine, 90 mM thymidine, 90 mM hypoxanthine,120 mM glycine, 5% Hy-soy peptone, and 100 mg/L insulin like growthfactor 1 (Rassmussen et al., Cytotechnology 28:31-42, 1998). For DHFRselection and methotrexate amplifications, the cells were cultured inthe same medium lacking thymidine hypoxanthine, and glycine. Formethotrexate selection, methotrexate (MTX; Lederle Laboratories, PearlRiver, N.Y.) is added to the selection medium at appropriateconcentrations. If neomycin selection is employed, 400 μg/ml of G418(Gibco, Grand Island, N.Y.) is included in the medium. The cells aretransfected using calcium phosphate transfection (Wigler et al. supra),or Lipofectamine™ transfection as recommended by the supplier (GibcoBRL, Gaithersburg, Md.). Lipofectamine™ Reagent is a commerciallyavailable reagent used to form lipid-nucleic acid complexes (orliposomes) which, when applied to cultured cells, facilitate uptake ofthe nucleic acid into the cells.

Example 2

[0066] This example describes a semi-quantitive polymerase chainreaction (PCR) technique that was used to confirm that the IL4R and DHFRmessages encoded by the plasmids described above were made and provideinformation on the relative levels of the various mRNAs. Cells weretransfected and cultured as described, and mRNA was obtained using anRNeasy total RNA isolation kit (Quiagen, Chatsworth, Calif.), andtreated with RNAse-free DNAse to diminish DNA contamination. Oligo-dTprimers were used to prepare the first strand cDNA; a control primer foractin was included to facilitate quantification.

[0067] The first strand was amplified and the amount of input RNAdetermined using a GeneAmp 5700 from PE Biosystems (Foster City,Calif.). Thirty cycles of PCR were performed and real-time quantitationof the PCR products was achieved using the double-stranded DNA bindingdye SYBR Green I (PE Biosystems, Foster City, Calif.). A standard curvewas prepared using known amounts of actin cDNA, IL-4R cDNA, and DHFRcDNA. The amount of cDNA in each sample was normalized using the amountof actin cDNA. The relative amounts of IL-4R and DHFR in each sample areshown in Table 2. TABLE 2 Construct IL-4R/Actin DHFR/Actin pBGH  4.2 7.4pMPLA 32.5 2.0

[0068] These data demonstrate that cells transfected with the alternatepolyadenylation vector have about 8 times as much IL-4R specific messageas the control, and the amount of DHFR is reduced 3.5-fold relative tothe control. This technique can be used to evaluate additionalpolyadenylation signals for use in the inventive expression vectors.

Example 3

[0069] This example describes an enzyme-linked immunosorbent assay(ELISA) that can be used to monitor production of recombinant proteins.The ELISA is well known in the art; adaptations of the techniquesdisclosed in Engvall et al., Immunochem. 8:871, 1971 and in U.S. Pat.No. 4,703,004 have been used to monitor production of variousrecombinant proteins. In this assay, a first antibody specific for aprotein of interest (usually a monoclonal antibody) is immobilized on asubstrate (most often, a 96-well microtiter plate), then a samplecontaining the protein is added and incubated. A series of dilutions ofa known concentration of the protein is also added and incubated, toyield a standard curve. After a wash step to remove unbound proteins andother materials, a second antibody to the protein is added. The secondantibody is directed against a different epitope of the protein, and maybe either a monoclonal antibody or a polyclonal antibody.

[0070] A conjugate reagent comprising an antibody that binds to thesecond antibody conjugated to an enzyme such as horse radish peroxidase(HRP) is added, either after a second wash step to remove unboundprotein, or at the same time the second antibody is added. Following asuitable incubation period, unbound conjugate reagent is removed bywashing, and a developing solution containing the substrate for theenzyme conjugate is added to the plate, causing color to develop. Theoptical density readings at the correct wavelength give numerical valuesfor each well. The values for the sample are compared with the standardcurve values, permitting levels of the desired protein to bequantitated.

[0071] To quantitate sIL-4R, an ELISA using two monoclonal antibodies(MAb) directed to different epitopes of IL-4R was developed. The firstMAb (referred to as M10) was adsorbed onto plates overnight, and theperoxidase (HRP) conjugated second antibody (referred to as HRP-M8) wasadded after a wash step.

Example 4

[0072] This example describes the transfection of CHO cells with variousconstructs and compares the production of sIL-4R by pools of transfectedcells. The various sIL-4R expression plasmids were transfected into CHOcells using Lipofectamine™. Cells were first selected for the DHFR+phenotype, then pooled and selected at different MTX concentrations.Pools of cells were grown for two to three days, then supernatant fluidharvested and analyzed by ELISA as described in Example 3, and specificproductivity (defined as μg of protein produced per day by 10⁴ cells)was determined. The results of a representative experiment are shown inTable 3 below. TABLE 3 Specific Cells/ml × % ELISA ProductivityConstruct 10⁶ Viable (μg protein) μg/10⁶ cells/day BGH, Control 1.98 940.3 0.12 SPA4, SV40 late 1.57 89 2.3 1.11 SPA6, SV40 early 2.42 91 3.21.10 MLPA 1.81 92 2.5 1.08 PY, Polyoma virus 2.4  93 0.4 0.14

[0073] These results demonstrated that the insertion of internal polyAsites in between a DNA encoding a desired recombinant protein and a DNAencoding a selectable marker can enhance expression of the desiredrecombinant protein from pools of transfected cells.

Example 5

[0074] This example illustrates the production of sIL-4R by pools oftransfected CHO cells over time. A high level of expression was stableover many passages. Four independent transfections with the MLPA plasmidwere performed substantially as described previously, and passaged over20 generations. Expression was monitored from each culture individually,and specific productivity results were averaged; the averages are shownin Table 4. TABLE 4 Passage Specific Productivity Standard number μg/10⁶cells/day deviation  5 1.22 0.41 10 1.26 0.39 15 1.18 0.55 18 1.09 0.4920 1.02 0.62

[0075] As can be seen in the data from Table 4, expression remainedstable over 20 passages. Cells from passage 20 from two of the poolswere then amplified in 5 nm methotrexate and monitored for IL4Rexpression; results are shown in Table 5. Amplified pools exhibitedincreased expression when compared to the unamplified pools. TABLE 5Passage Specific Productivity Specific Productivity number Pool #1 Pool#2 27 1.95 1.37 29 2.10 1.58 33 2.08 1.42

Example 6

[0076] This example illustrates the effect of internal polyA sites onclones of cells derived from transfected pools. BGH, SPA4 and SPA6 cellswere cloned by limiting dilution in the presence of MTX. Severalcolonies were picked and screened for specific productivity of sIL-4R asdescribed for the pools. Results are shown in Table 6. TABLE 6 SpecificMTX % ELISA Productivity Construct Clone # Concentration Cells/ml Viable(μg protein) μg/10⁶ cells/day BGH 3 200 1.84 69 14.6 4.25 SPA4 2 50 2.2689 16.2 3.99 SPA6 10 100 1.82 94 1.6 0.47 SPA6 11 100 1.46 83 7 2.44SPA6 13 100 0.96 67 9.3 4.40 SPA6 16 100 1.02 73 0 0

[0077] These results demonstrate that the clones picked from the poolstransfected with expression vectors comprising an internal polyA sitecan express high levels of the desired recombinant protein. For thepurposes of producing large amounts of recombinant protein for use as apharmaceutical, clones are often reamplified in methotrexate. In orderto evaluate the effect of an internal polyA site on the reamplificationprocess, clone 2 from the SPA4 pool was reamplified by culturing thecells for several passages in increasing concentrations of methotrexate.Once the cells had recovered from the methotrexate amplification withviabilities of about 90%, the specific productivity was determined byculturing the cells for two to three days, harvesting the supernatantfluid, and assaying the supernatant fluid for IL-4R by ELISA; resultsare shown in Table 7. TABLE 7 Specific Methotrexate ELISA ProductivityConstruct Concentration Cells/ml % Viable (μg protein) μg/10⁶ cells/daySPA4-2  50 nM 2.32 94 18.7 4.58 SPA4-2 100 nM 1.66 91 21.1 6.83 SPA4-2150 nM 1.95 90 27.7 7.86 SPA4-2 200 nM 1.93 91 28.8 8.24

[0078] These results demonstrated that clones of cells transfected withexpression vectors comprising an internal polyA site can be reamplified,and will be expected to evince higher specific productivity.

Example 7

[0079] This example describes the preparation of several expressionvectors for the expression of recombinant proteins. An expression vectorencoding a marker protein (secreted alkaline phosphatase or SEAP; Bergeret al., Gene 66:1, 1988) is prepared substantially as describedpreviously, using the MLPA polyA site internally; a polyA site otherthan BGH may be used as the terminal polyA site. Several changes aremade to the IRES sequence within the expression vectors. As discussed inDavies and Kaufman (J. Virology 66:1924; 1992), the efficiency oftranslation of a second gene can be manipulated by altering the sequenceof the IRES at or near the junction of the IRES with the second gene, inthis case, DHFR. Table 8 depicts the nucleotide sequence added to theIRES; the first base indicated in the Table is directly after nucleotide566 of the EMCV IRES (SEQ ID NO:7). Translational start sites (ATG) areunderlined; the 3′ATG is the first ATG of muDHFR. TABLE 8 Construct DNASequence at IIRES DHFR junction IX-312 ATTGCTCGAGATCCGTGCCATCATG (SEQ IDNO:8) IXED-1 ATGATAATATG (SEQ ID NO:9) IXED-3 ATGATAATATGGCCACAACCATG(SEQ ID NO:10)

[0080] Appending the nucleotide sequences to the IRES will modulateexpression of DHFR sufficiently to increase the percentage of cellstransfected without significantly decreasing the levels of the desiredrecombinant protein. The vectors (including control vectors) are used instandard transfections to prepare transfected cells expressing SEAPsubstantially as described herein. Expression levels of the markerprotein, SEAP, are determined using a quantitative assay such as thatavailable from CLONTECH Laboratories (Palo Alto, Calif., USA; Yang etal., Biotechniques 2:1110, 1997).

1 10 1 222 DNA SV40 1 atccagacat gataagatac attgatgagt ttggacaaaccacaactaga atgcagtgaa 60 aaaaatgctt tatttgtgaa atttgtgatg ctattgctttatttgtaacc attataagct 120 gcaataaaca agttcaacaa caattgcatt cattttatgtttcaggttca gggggaggtg 180 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gt222 2 285 DNA Bovine 2 aattgtctag agctcgctga tcagcctcga ctgtgccttctagttgccag ccatctgttg 60 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgccactcccact gtcctttcct 120 aataaaatga ggaaattgca tcgcattgtc tgagtaggtgtcattctatt ctggggggtg 180 gggtggggca ggacagcaag ggggaggatt gggaagacaatagcaggcat gctggggatg 240 cggtgggctc tatggcttct gaggcggaaa gaaccagctggggca 285 3 222 DNA SV40 3 accacatttg tagaggtttt acttgcttta aaaaacctcccacacctccc cctgaacctg 60 aaacataaaa tgaatgcaat tgttgttgaa cttgtttattgcagcttata atggttacaa 120 ataaagcaat agcatcacaa atttcacaaa taaagcatttttttcactgc attctagttg 180 tggtttgtcc aaactcatca atgtatctta tcatgtctgg at222 4 19 RNA RNA recognition sequence 4 acaugaggau uacccaugu 19 5 2478DNA Homo sapiens CDS (1)..(2478) mat_peptide (76)..() sig_peptide(1)..(75) 5 atg ggg tgg ctt tgc tct ggg ctc ctg ttc cct gtg agc tgc ctggtc 48 Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val-25 -20 -15 -10 ctg ctg cag gtg gca agc tct ggg aac atg aag gtc ttg caggag ccc 96 Leu Leu Gln Val Ala Ser Ser Gly Asn Met Lys Val Leu Gln GluPro -5 -1 1 5 acc tgc gtc tcc gac tac atg agc atc tct act tgc gag tggaag atg 144 Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp LysMet 10 15 20 aat ggt ccc acc aat tgc agc acc gag ctc cgc ctg ttg tac cagctg 192 Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu25 30 35 gtt ttt ctg ctc tcc gaa gcc cac acg tgt atc cct gag aac aac gga240 Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly 4045 50 55 ggc gcg ggg tgc gtg tgc cac ctg ctc atg gat gac gtg gtc agt gcg288 Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala 6065 70 gat aac tat aca ctg gac ctg tgg gct ggg cag cag ctg ctg tgg aag336 Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys 7580 85 ggc tcc ttc aag ccc agc gag cat gtg aaa ccc agg gcc cca gga aac384 Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn 9095 100 ctg aca gtt cac acc aat gtc tcc gac act ctg ctg ctg acc tgg agc432 Leu Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser 105110 115 aac ccg tat ccc cct gac aat tac ctg tat aat cat ctc acc tat gca480 Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala 120125 130 135 gtc aac att tgg agt gaa aac gac ccg gca gat ttc aga atc tataac 528 Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn140 145 150 gtg acc tac cta gaa ccc tcc ctc cgc atc gca gcc agc acc ctgaag 576 Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys155 160 165 tct ggg att tcc tac agg gca cgg gtg agg gcc tgg gct cag tgctat 624 Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr170 175 180 aac acc acc tgg agt gag tgg agc ccc agc acc aag tgg cac aactcc 672 Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser185 190 195 tac agg gag ccc ttc gag cag cac ctc ctg ctg ggc gtc agc gtttcc 720 Tyr Arg Glu Pro Phe Glu Gln His Leu Leu Leu Gly Val Ser Val Ser200 205 210 215 tgc att gtc atc ctg gcc gtc tgc ctg ttg tgc tat gtc agcatc acc 768 Cys Ile Val Ile Leu Ala Val Cys Leu Leu Cys Tyr Val Ser IleThr 220 225 230 aag att aag aaa gaa tgg tgg gat cag att ccc aac cca gcccgc agc 816 Lys Ile Lys Lys Glu Trp Trp Asp Gln Ile Pro Asn Pro Ala ArgSer 235 240 245 cgc ctc gtg gct ata ata atc cag gat gct cag ggg tca cagtgg gag 864 Arg Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Gly Ser Gln TrpGlu 250 255 260 aag cgg tcc cga ggc cag gaa cca gcc aag tgc cca cac tggaag aat 912 Lys Arg Ser Arg Gly Gln Glu Pro Ala Lys Cys Pro His Trp LysAsn 265 270 275 tgt ctt acc aag ctc ttg ccc tgt ttt ctg gag cac aac atgaaa agg 960 Cys Leu Thr Lys Leu Leu Pro Cys Phe Leu Glu His Asn Met LysArg 280 285 290 295 gat gaa gat cct cac aag gct gcc aaa gag atg cct ttccag ggc tct 1008 Asp Glu Asp Pro His Lys Ala Ala Lys Glu Met Pro Phe GlnGly Ser 300 305 310 gga aaa tca gca tgg tgc cca gtg gag atc agc aag acagtc ctc tgg 1056 Gly Lys Ser Ala Trp Cys Pro Val Glu Ile Ser Lys Thr ValLeu Trp 315 320 325 cca gag agc atc agc gtg gtg cga tgt gtg gag ttg tttgag gcc ccg 1104 Pro Glu Ser Ile Ser Val Val Arg Cys Val Glu Leu Phe GluAla Pro 330 335 340 gtg gag tgt gag gag gag gag gag gta gag gaa gaa aaaggg agc ttc 1152 Val Glu Cys Glu Glu Glu Glu Glu Val Glu Glu Glu Lys GlySer Phe 345 350 355 tgt gca tcg cct gag agc agc agg gat gac ttc cag gaggga agg gag 1200 Cys Ala Ser Pro Glu Ser Ser Arg Asp Asp Phe Gln Glu GlyArg Glu 360 365 370 375 ggc att gtg gcc cgg cta aca gag agc ctg ttc ctggac ctg ctc gga 1248 Gly Ile Val Ala Arg Leu Thr Glu Ser Leu Phe Leu AspLeu Leu Gly 380 385 390 gag gag aat ggg ggc ttt tgc cag cag gac atg ggggag tca tgc ctt 1296 Glu Glu Asn Gly Gly Phe Cys Gln Gln Asp Met Gly GluSer Cys Leu 395 400 405 ctt cca cct tcg gga agt acg agt gct cac atg ccctgg gat gag ttc 1344 Leu Pro Pro Ser Gly Ser Thr Ser Ala His Met Pro TrpAsp Glu Phe 410 415 420 cca agt gca ggg ccc aag gag gca cct ccc tgg ggcaag gag cag cct 1392 Pro Ser Ala Gly Pro Lys Glu Ala Pro Pro Trp Gly LysGlu Gln Pro 425 430 435 ctc cac ctg gag cca agt cct cct gcc agc ccg acccag agt cca gac 1440 Leu His Leu Glu Pro Ser Pro Pro Ala Ser Pro Thr GlnSer Pro Asp 440 445 450 455 aac ctg act tgc aca gag acg ccc ctc gtc atcgca ggc aac cct gct 1488 Asn Leu Thr Cys Thr Glu Thr Pro Leu Val Ile AlaGly Asn Pro Ala 460 465 470 tac cgc agc ttc agc aac tcc ctg agc cag tcaccg tgt ccc aga gag 1536 Tyr Arg Ser Phe Ser Asn Ser Leu Ser Gln Ser ProCys Pro Arg Glu 475 480 485 ctg ggt cca gac cca ctg ctg gcc aga cac ctggag gaa gta gaa ccc 1584 Leu Gly Pro Asp Pro Leu Leu Ala Arg His Leu GluGlu Val Glu Pro 490 495 500 gag atg ccc tgt gtc ccc cag ctc tct gag ccaacc act gtg ccc caa 1632 Glu Met Pro Cys Val Pro Gln Leu Ser Glu Pro ThrThr Val Pro Gln 505 510 515 cct gag cca gaa acc tgg gag cag atc ctc cgccga aat gtc ctc cag 1680 Pro Glu Pro Glu Thr Trp Glu Gln Ile Leu Arg ArgAsn Val Leu Gln 520 525 530 535 cat ggg gca gct gca gcc ccc gtc tcg gccccc acc agt ggc tat cag 1728 His Gly Ala Ala Ala Ala Pro Val Ser Ala ProThr Ser Gly Tyr Gln 540 545 550 gag ttt gta cat gcg gtg gag cag ggt ggcacc cag gcc agt gcg gtg 1776 Glu Phe Val His Ala Val Glu Gln Gly Gly ThrGln Ala Ser Ala Val 555 560 565 gtg ggc ttg ggt ccc cca gga gag gct ggttac aag gcc ttc tca agc 1824 Val Gly Leu Gly Pro Pro Gly Glu Ala Gly TyrLys Ala Phe Ser Ser 570 575 580 ctg ctt gcc agc agt gct gtg tcc cca gagaaa tgt ggg ttt ggg gct 1872 Leu Leu Ala Ser Ser Ala Val Ser Pro Glu LysCys Gly Phe Gly Ala 585 590 595 agc agt ggg gaa gag ggg tat aag cct ttccaa gac ctc att cct ggc 1920 Ser Ser Gly Glu Glu Gly Tyr Lys Pro Phe GlnAsp Leu Ile Pro Gly 600 605 610 615 tgc cct ggg gac cct gcc cca gtc cctgtc ccc ttg ttc acc ttt gga 1968 Cys Pro Gly Asp Pro Ala Pro Val Pro ValPro Leu Phe Thr Phe Gly 620 625 630 ctg gac agg gag cca cct cgc agt ccgcag agc tca cat ctc cca agc 2016 Leu Asp Arg Glu Pro Pro Arg Ser Pro GlnSer Ser His Leu Pro Ser 635 640 645 agc tcc cca gag cac ctg ggt ctg gagccg ggg gaa aag gta gag gac 2064 Ser Ser Pro Glu His Leu Gly Leu Glu ProGly Glu Lys Val Glu Asp 650 655 660 atg cca aag ccc cca ctt ccc cag gagcag gcc aca gac ccc ctt gtg 2112 Met Pro Lys Pro Pro Leu Pro Gln Glu GlnAla Thr Asp Pro Leu Val 665 670 675 gac agc ctg ggc agt ggc att gtc tactca gcc ctt acc tgc cac ctg 2160 Asp Ser Leu Gly Ser Gly Ile Val Tyr SerAla Leu Thr Cys His Leu 680 685 690 695 tgc ggc cac ctg aaa cag tgt catggc cag gag gat ggt ggc cag acc 2208 Cys Gly His Leu Lys Gln Cys His GlyGln Glu Asp Gly Gly Gln Thr 700 705 710 cct gtc atg gcc agt cct tgc tgtggc tgc tgc tgt gga gac agg tcc 2256 Pro Val Met Ala Ser Pro Cys Cys GlyCys Cys Cys Gly Asp Arg Ser 715 720 725 tcg ccc cct aca acc ccc ctg agggcc cca gac ccc tct cca ggt ggg 2304 Ser Pro Pro Thr Thr Pro Leu Arg AlaPro Asp Pro Ser Pro Gly Gly 730 735 740 gtt cca ctg gag gcc agt ctg tgtccg gcc tcc ctg gca ccc tcg ggc 2352 Val Pro Leu Glu Ala Ser Leu Cys ProAla Ser Leu Ala Pro Ser Gly 745 750 755 atc tca gag aag agt aaa tcc tcatca tcc ttc cat cct gcc cct ggc 2400 Ile Ser Glu Lys Ser Lys Ser Ser SerSer Phe His Pro Ala Pro Gly 760 765 770 775 aat gct cag agc tca agc cagacc ccc aaa atc gtg aac ttt gtc tcc 2448 Asn Ala Gln Ser Ser Ser Gln ThrPro Lys Ile Val Asn Phe Val Ser 780 785 790 gtg gga ccc aca tac atg agggtc tct tat 2478 Val Gly Pro Thr Tyr Met Arg Val Ser Tyr 795 800 6 826PRT Homo sapiens 6 Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val SerCys Leu Val -25 -20 -15 -10 Leu Leu Gln Val Ala Ser Ser Gly Asn Met LysVal Leu Gln Glu Pro -5 -1 1 5 Thr Cys Val Ser Asp Tyr Met Ser Ile SerThr Cys Glu Trp Lys Met 10 15 20 Asn Gly Pro Thr Asn Cys Ser Thr Glu LeuArg Leu Leu Tyr Gln Leu 25 30 35 Val Phe Leu Leu Ser Glu Ala His Thr CysIle Pro Glu Asn Asn Gly 40 45 50 55 Gly Ala Gly Cys Val Cys His Leu LeuMet Asp Asp Val Val Ser Ala 60 65 70 Asp Asn Tyr Thr Leu Asp Leu Trp AlaGly Gln Gln Leu Leu Trp Lys 75 80 85 Gly Ser Phe Lys Pro Ser Glu His ValLys Pro Arg Ala Pro Gly Asn 90 95 100 Leu Thr Val His Thr Asn Val SerAsp Thr Leu Leu Leu Thr Trp Ser 105 110 115 Asn Pro Tyr Pro Pro Asp AsnTyr Leu Tyr Asn His Leu Thr Tyr Ala 120 125 130 135 Val Asn Ile Trp SerGlu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn 140 145 150 Val Thr Tyr LeuGlu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys 155 160 165 Ser Gly IleSer Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr 170 175 180 Asn ThrThr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser 185 190 195 TyrArg Glu Pro Phe Glu Gln His Leu Leu Leu Gly Val Ser Val Ser 200 205 210215 Cys Ile Val Ile Leu Ala Val Cys Leu Leu Cys Tyr Val Ser Ile Thr 220225 230 Lys Ile Lys Lys Glu Trp Trp Asp Gln Ile Pro Asn Pro Ala Arg Ser235 240 245 Arg Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Gly Ser Gln TrpGlu 250 255 260 Lys Arg Ser Arg Gly Gln Glu Pro Ala Lys Cys Pro His TrpLys Asn 265 270 275 Cys Leu Thr Lys Leu Leu Pro Cys Phe Leu Glu His AsnMet Lys Arg 280 285 290 295 Asp Glu Asp Pro His Lys Ala Ala Lys Glu MetPro Phe Gln Gly Ser 300 305 310 Gly Lys Ser Ala Trp Cys Pro Val Glu IleSer Lys Thr Val Leu Trp 315 320 325 Pro Glu Ser Ile Ser Val Val Arg CysVal Glu Leu Phe Glu Ala Pro 330 335 340 Val Glu Cys Glu Glu Glu Glu GluVal Glu Glu Glu Lys Gly Ser Phe 345 350 355 Cys Ala Ser Pro Glu Ser SerArg Asp Asp Phe Gln Glu Gly Arg Glu 360 365 370 375 Gly Ile Val Ala ArgLeu Thr Glu Ser Leu Phe Leu Asp Leu Leu Gly 380 385 390 Glu Glu Asn GlyGly Phe Cys Gln Gln Asp Met Gly Glu Ser Cys Leu 395 400 405 Leu Pro ProSer Gly Ser Thr Ser Ala His Met Pro Trp Asp Glu Phe 410 415 420 Pro SerAla Gly Pro Lys Glu Ala Pro Pro Trp Gly Lys Glu Gln Pro 425 430 435 LeuHis Leu Glu Pro Ser Pro Pro Ala Ser Pro Thr Gln Ser Pro Asp 440 445 450455 Asn Leu Thr Cys Thr Glu Thr Pro Leu Val Ile Ala Gly Asn Pro Ala 460465 470 Tyr Arg Ser Phe Ser Asn Ser Leu Ser Gln Ser Pro Cys Pro Arg Glu475 480 485 Leu Gly Pro Asp Pro Leu Leu Ala Arg His Leu Glu Glu Val GluPro 490 495 500 Glu Met Pro Cys Val Pro Gln Leu Ser Glu Pro Thr Thr ValPro Gln 505 510 515 Pro Glu Pro Glu Thr Trp Glu Gln Ile Leu Arg Arg AsnVal Leu Gln 520 525 530 535 His Gly Ala Ala Ala Ala Pro Val Ser Ala ProThr Ser Gly Tyr Gln 540 545 550 Glu Phe Val His Ala Val Glu Gln Gly GlyThr Gln Ala Ser Ala Val 555 560 565 Val Gly Leu Gly Pro Pro Gly Glu AlaGly Tyr Lys Ala Phe Ser Ser 570 575 580 Leu Leu Ala Ser Ser Ala Val SerPro Glu Lys Cys Gly Phe Gly Ala 585 590 595 Ser Ser Gly Glu Glu Gly TyrLys Pro Phe Gln Asp Leu Ile Pro Gly 600 605 610 615 Cys Pro Gly Asp ProAla Pro Val Pro Val Pro Leu Phe Thr Phe Gly 620 625 630 Leu Asp Arg GluPro Pro Arg Ser Pro Gln Ser Ser His Leu Pro Ser 635 640 645 Ser Ser ProGlu His Leu Gly Leu Glu Pro Gly Glu Lys Val Glu Asp 650 655 660 Met ProLys Pro Pro Leu Pro Gln Glu Gln Ala Thr Asp Pro Leu Val 665 670 675 AspSer Leu Gly Ser Gly Ile Val Tyr Ser Ala Leu Thr Cys His Leu 680 685 690695 Cys Gly His Leu Lys Gln Cys His Gly Gln Glu Asp Gly Gly Gln Thr 700705 710 Pro Val Met Ala Ser Pro Cys Cys Gly Cys Cys Cys Gly Asp Arg Ser715 720 725 Ser Pro Pro Thr Thr Pro Leu Arg Ala Pro Asp Pro Ser Pro GlyGly 730 735 740 Val Pro Leu Glu Ala Ser Leu Cys Pro Ala Ser Leu Ala ProSer Gly 745 750 755 Ile Ser Glu Lys Ser Lys Ser Ser Ser Ser Phe His ProAla Pro Gly 760 765 770 775 Asn Ala Gln Ser Ser Ser Gln Thr Pro Lys IleVal Asn Phe Val Ser 780 785 790 Val Gly Pro Thr Tyr Met Arg Val Ser Tyr795 800 7 566 DNA EMCV 7 cccctctccc tccccccccc ctaacgttac tggccgaagccgcttggaat aaggccggtg 60 tgcgtttgtc tatatgttat tttccaccat attgccgtcttttggcaatg tgagggcccg 120 gaaacctggc cctgtcttct tgacgagcat tcctaggggtctttcccctc tcgccaaagg 180 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcctctggaagctt cttgaagaca 240 aacaacgtct gtagcgaccc tttgcaggca gcggaaccccccacctggcg acaggtgcct 300 ctgcggccaa aagccacgtg tataagatac acctgcaaaggcggcacaac cccagtgcca 360 cgttgtgagt tggatagttg tggaaagagt caaatggctctcctcaagcg tattcaacaa 420 ggggctgaag gatgcccaga aggtacccca ttgtatgggatctgatctgg ggcctcggtg 480 cacatgcttt acatgtgttt agtcgaggtt aaaaaacgtctaggcccccc gaaccacggg 540 gacgtggttt tcctttgaaa aacacg 566 8 25 DNA EMCV8 attgctcgag atccgtgcca tcatg 25 9 11 DNA EMCV 9 atgataatat g 11 10 23DNA EMCV 10 atgataatat ggccacaacc atg 23

What is claimed is:
 1. An expression vector comprising, in the followingorder, a promoter sequence, a first coding sequence, a polyadenylationsite, and a second coding sequence, wherein no promoter sequence occursbetween the internal polyadenylation site and the second codingsequence.
 2. The expression vector of claim 1, wherein the second codingsequence encodes a selectable marker.
 3. The expression vector of claim2, wherein the selectable marker is dihydrofolate reductase.
 4. Theexpression vector of claim 1 wherein the internal polyadenylation siteis selected from the group consisting of SV40 late polyadenylation site(SEQ ID NO:1), SV40 early polyadenylation site (SEQ ID NO:3), and amutant SV40 late polyadenylation site consisting essentially ofnucleotides 80 through 222 of SEQ ID NO:1.
 5. The expression vector ofclaim 2 wherein the internal polyadenylation site is selected from thegroup consisting of SV40 late polyadenylation site (SEQ ID NO:1), SV40early polyadenylation site (SEQ ID NO:3), and a mutant SV40 latepolyadenylation site consisting essentially of nucleotides 80 through222 of SEQ ID NO:1.
 6. The expression vector of claim 3 wherein theinternal polyadenylation site is selected from the group consisting ofSV40 late polyadenylation site (SEQ ID NO:1), SV40 early polyadenylationsite (SEQ ID NO:3), and a mutant SV40 late polyadenylation siteconsisting essentially of nucleotides 80 through 222 of SEQ ID NO:1. 7.The expression vector of claim 1 wherein an internal ribosome entry site(IRES) is inserted between the DNA encoding the internal polyadenylationsite and the DNA encoding the selectable marker such that the IRES isoperably linked to the selectable marker.
 8. The expression vector ofclaim 2 wherein an internal ribosome entry site (IRES) is insertedbetween the DNA encoding the internal polyadenylation site and the DNAencoding the selectable marker such that the IRES is operably linked tothe selectable marker.
 9. The expression vector of claim 3 wherein aninternal ribosome entry site (IRES) is inserted between the DNA encodingthe internal polyadenylation site and the DNA encoding the selectablemarker such that the IRES is operably linked to the selectable marker.10. The expression vector of claim 4 wherein an internal ribosome entrysite (IRES) is inserted between the DNA encoding the internalpolyadenylation site and the DNA encoding the selectable marker suchthat the IRES is operably linked to the selectable marker.
 11. Theexpression vector of claim 5 wherein an internal ribosome entry site(IRES) is inserted between the DNA encoding the internal polyadenylationsite and the DNA encoding the selectable marker such that the IRES isoperably linked to the selectable marker.
 12. The expression vector ofclaim 6 wherein an internal ribosome entry site (IRES) is insertedbetween the DNA encoding the internal polyadenylation site and the DNAencoding the selectable marker such that the IRES is operably linked tothe selectable marker.
 13. A stable pools of cells transfected with anexpression vector according to claim
 7. 14. A stable pools of cellstransfected with an expression vector according to claim
 8. 15. A stablepools of cells transfected with an expression vector according to claim9.
 16. A stable pools of cells transfected with an expression vectoraccording to claim
 10. 17. A stable pools of cells transfected with anexpression vector according to claim
 11. 18. A stable pools of cellstransfected with an expression vector according to claim
 12. 19. A cellline cloned from the pool of claim
 13. 20. A cell line cloned from thepool of claim
 14. 21. A cell line cloned from the pool of claim
 15. 22.A cell line cloned from the pool of claim
 16. 23. A cell line clonedfrom the pool of claim
 17. 24. A cell line cloned from the pool of claim18.
 25. A method for obtaining a recombinant protein, comprisingculturing a stable pool of cells according to claim 13 under conditionspromoting expression of the protein, and recovering the protein.
 26. Amethod for obtaining a recombinant protein, comprising culturing astable pool of cells according to claim 14 under conditions promotingexpression of the protein, and recovering the protein.
 27. A method forobtaining a recombinant protein, comprising culturing a stable pool ofcells according to claim 15 under conditions promoting expression of theprotein, and recovering the protein.
 28. A method for obtaining arecombinant protein, comprising culturing a stable pool of cellsaccording to claim 16 under conditions promoting expression of theprotein, and recovering the protein.
 29. A method for obtaining arecombinant protein, comprising culturing a stable pool of cellsaccording to claim 17 under conditions promoting expression of theprotein, and recovering the protein.
 30. A method for obtaining arecombinant protein, comprising culturing a stable pool of cellsaccording to claim 18 under conditions promoting expression of theprotein, and recovering the protein.
 31. A method for obtaining arecombinant protein, comprising culturing a cell line according to claim19 under conditions promoting expression of the protein, and recoveringthe protein.
 32. A method for obtaining a recombinant protein,comprising culturing a cell line according to claim 20 under conditionspromoting expression of the protein, and recovering the protein.
 33. Amethod for obtaining a recombinant protein, comprising culturing a cellline according to claim 21 under conditions promoting expression of theprotein, and recovering the protein.
 34. A method for obtaining arecombinant protein, comprising culturing a cell line according to claim22 under conditions promoting expression of the protein, and recoveringthe protein.
 35. A method for obtaining a recombinant protein,comprising culturing a cell line according to claim 23 under conditionspromoting expression of the protein, and recovering the protein.
 36. Amethod for obtaining a recombinant protein, comprising culturing a cellline according to claim 24 under conditions promoting expression of theprotein, and recovering the protein.
 37. The expression vector of claim1, which further comprises a second polyadenylation site following thesecond coding sequence and operably linked thereto.
 38. An expressionvector comprising, in the following order, a promoter sequence, a firstcoding sequence, a polyadenylation site consisting essentially ofnucleotides 80 through 222 of SEQ ID NO:1, and a second coding sequence,wherein no promoter sequence occurs between the internal polyadenylationsite and the second coding sequence.
 39. A stable pool of mammaliancells transfected with an expression vector according to claim
 38. 40. Acell line cloned from the pool of claim
 39. 41. A method for obtaining arecombinant protein, comprising culturing a stable pool of cellsaccording to claim 39 under conditions promoting expression of theprotein, and recovering the protein.
 42. A method for obtaining arecombinant protein, comprising culturing a cell line according to claim40 under conditions promoting expression of the protein, and recoveringthe protein.
 43. A mammalian host cell containing an expression vectoraccording to claim
 7. 44. A mammalian host cell containing an expressionvector according to claim
 8. 45. A mammalian host cell containing anexpression vector according to claim
 9. 46. A mammalian host cellcontaining an expression vector according to claim
 10. 47. A mammalianhost cell containing an expression vector according to claim
 11. 48. Amammalian host cell containing an expression vector according to claim36.
 49. The expression vector of claim 38, which further comprises asecond polyadenylation site following the second coding sequence andoperably linked thereto.